Luminescent microscopy is a traditional field of application of light microscopy used to analyze biological preparations. In the process, certain dyes (so-called phosphors or fluorophores) are used for the specific labeling of samples, e.g. of cell parts. As mentioned, a sample is illuminated by means of illumination radiation representing excitation radiation and the luminescent radiation induced in this way is recorded with suitable detectors. For this purpose, a dichroic beam splitter combined with block filters are normally provided in the microscope which split the luminescent radiation from the excitation radiation and allow an independent observation. This approach makes it possible to depict individual, differently stained cell parts under the microscope. Of course, a plurality of parts of a single preparation can also be stained simultaneously with different dyes which specifically adhere to different structures of the preparation. This method is referred to as multiple luminescence. Furthermore, it is also possible to measure samples which are luminescent per se, i.e. without the addition of dyes.
As is the rule, luminescence here is the generic term for phosphorescence and fluorescence, meaning that it includes both processes. Whenever the term fluorescence is mentioned in this document, it is deemed a part or aspect of something taken as representative of the whole rather than a limitation.
Laser scanning microscopes (in short LSM) are another known device used for analyzing samples. Of a three-dimensionally illuminated image, they only depict the plane located within the focal plane of the lens by means of a confocal detection array (referred to as a confocal LSM) or a non-linear sample interaction (so-called multiphoton microscopy). An optical cross section is obtained and the documentation of a plurality of optical cross sections in different depths of the sample subsequently makes it possible to generate a three-dimensional image of the sample by means of a suitable data processing device which is composed of the different optical cross sections. Consequently, laser scanning microscopy is suitable for analyzing thick preparations.
Of course, a combination of luminescence microscopy and laser scanning microscopy is also possible, in which a luminescent sample is depicted at different depth planes by means of an LSM.
In principle, the optical resolution of a light microscope, including the one of an LSM is diffraction-limited as a result of the laws of physics. Special illumination configurations for the optimal resolution within these limits are known, such as for example the 4Pi arrangement or arrangements with standing-wave fields. This helps considerably improve the resolution, in particular in an axial direction compared to a traditional LSM. The resolution can be further increased to a factor of up to 10 compared to a diffraction-limited confocal LSM by means of non-linear depopulation processes. Such a method is disclosed for example in U.S. Pat. No. 5,866,911. Different approaches are known for the depopulation processes, such as described for example in DE 4416558 C2, U.S. Pat. No. 6,633,432 or DE 10325460 A1.
A further method for the resolution enhancement is discussed in EP 11579297 B1. In it, non-linear 2
processes are to be used by means of structured illumination. The published document mentions the fluorescence saturation as non-linearity. The described method claims to realize a displacement of the object space spectrum relative to the transmission function of the optical system by way of structured illumination. Specifically, the displacement of the spectrum means that object space frequencies V0 are transmitted at a spatial frequency of V0-Vm, where Vm is the frequency of the structured illumination. At a given spatial frequency maximally transmissible by the system, this enables the transfer of spatial frequencies of the object exceeding the maximum frequency of the transmission function by the displacement frequency Vm. This approach requires a reconstruction algorithm for the image generation and the utilization of several images for one picture. Furthermore, it is considered a disadvantage of this method that the sample is unnecessarily exposed to radiation outside the detected focus, because the required structured illumination covers the entire sample volume. Moreover, this method can currently not be used for thick samples, because extra-focally excited fluorescence also reaches the detector as background signal and hence drastically reduces the dynamic range of the detected radiation.
A method that achieves a resolution beyond the diffraction limit independently of laser scanning microscopy is disclosed in WO 2006127692 and DE 102006021317. This method abbreviated with the acronym PALM (Photo Activated Light Microscopy) uses a marker substance which can be activated by means of an optical activation signal. The marker substance can only be excited to emit certain fluorescent radiation by means of induction radiation in the activated state. Non-activated molecules of the marker substance will not emit any or at least no noticeable fluorescent radiation even after irradiation with excitation radiation. This means that the activation radiation switches the marker substance into a state in which it can be excited to emit fluorescence. Other types of activation, e.g. of thermal nature, are possible as well. Therefore, this is generally referred to as a switching signal. In the PALM method, the switching signal is applied in such a way that at least a certain ratio of activated marker molecules are spaced apart from adjacent activated molecules such that they are separated as measured by the optical resolution of the microscopy or can be separated retroactively. In other words, the activated molecules are at least for the most part isolated. After exposure to luminescent radiation, the center of their limited resolution-related radiation distribution is determined for said isolated molecules and this determination is used to calculate the position of the molecules with greater accuracy than the optical representation actually allows. Said enhanced resolution by way of mathematically calculated determination of the center of the diffraction distribution is referred to as “superresolution” in the English technical literature. It requires that at least some of the activated marker molecules in a sample are distinguishable, i.e. isolated with the optical resolution used to detect the luminescent radiation. If this is the case, the location information can then be achieved with increased resolution for these molecules.
To isolate individual marker molecules, the PALM method is based on the fact that the probability with which a marker molecule is activated after receiving the switching signal with a given intensity, e.g. a photon of the activation radiation, is identical for all molecules. In other words, the intensity of the switching signal and hence the number of photons striking a unit of area of the sample can be used to make sure that the probability of activating marker molecules present in a given area of the sample is so low that there are enough areas in which only distinguishable marker molecules emit fluorescent radiation within the optical resolution. The result of an appropriate selection of the intensity, e.g. the photon density of the switching signal, is that as much as possible only marker molecules are activated which are isolated relative to the optical resolution and subsequently emit fluorescent radiation. Next, the center of the diffraction-related intensity distribution and hence the position of the marker molecule is mathematically calculated with increased resolution for these isolated molecules. To image the entire sample, the isolation of the marker molecules of the subquantity by way of introducing activation radiation, subsequent excitation and fluorescent radiation imaging is repeated for as long as until as many marker molecules as possible were contained in one subquantity and isolated within the resolution of the image at once.
In the process, the advantage of the PALM method is that no high local resolution is required either for the activation or for the excitation. Instead, both the activation and the excitation can be achieved with wide-field illumination.
As a result, the marker molecules are statistically activated in subquantities by means of suitable selection of the intensity of the activation radiation. For this reason, a plurality of individual pictures need to be evaluated for the generation of a complete image of a sample in which the positions of all marker molecules can be calculated mathematically, e.g. by means of a resolution beyond the diffraction limit. This can concern up to 10,000 individual pictures. As a result, large data quantities are processed and the measurement takes a commensurate amount of time. The acquisition of a complete image alone takes several minutes which is essentially defined by the read-out rate of the camera used. The position of the molecules in the individual pictures is determined by means of complex mathematical procedures such as they are for instance described in Egner et al., Biophysical Journal, p. 3285-3290, volume 93, November 2007. The processing of all individual pictures and the composition into one complete high-resolution image, i.e. an image in which the locations of the marker molecules are illustrated with a resolution beyond the diffraction limit, typically takes one to two hours.